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Makara Journal of Science Makara Journal of Science
Volume 27
Issue 4
December
Article 4
12-10-2023
Enzymatic Screening and Genotypic Characterization of Enzymatic Screening and Genotypic Characterization of
Thermophilic Bacteria from the Hot Springs of Sarawak, Malaysia Thermophilic Bacteria from the Hot Springs of Sarawak, Malaysia
Seng Chiew Toh
Department of Animal Sciences and Fishery, Faculty of Agricultural and Forestry Sciences, Universiti Putra
Malaysia Bintulu Sarawak, Bintulu 97008, Malaysia
Samuel Lihan
Institute of Biodiversity and Environmental Conservation, Universiti Malaysia Sarawak, Kota Samarahan
94300, Malaysia
Sui Sien Leong
Universiti Putra Malaysia Bintulu Sarawak Campus
, leongsuisien@upm.edu.my
Azizul Hakim Lahuri
Department of Science and Technology, Faculty of Humanities, Management and Science, Universiti Putra
Malaysia Bintulu Sarawak, Bintulu 97008, Malaysia
Wai Cheong Woon
English Unit (CALC), Universiti Putra Malaysia Bintulu Sarawak, Bintulu 97008, Malaysia
See next page for additional authors
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Recommended Citation Recommended Citation
Toh, Seng Chiew; Lihan, Samuel; Leong, Sui Sien; Lahuri, Azizul Hakim; Woon, Wai Cheong; and Ng, Wing
Woh (2023) "Enzymatic Screening and Genotypic Characterization of Thermophilic Bacteria from the Hot
Springs of Sarawak, Malaysia,"
Makara Journal of Science
: Vol. 27: Iss. 4, Article 4.
DOI: 10.7454/mss.v27i4.1449
Available at: https://scholarhub.ui.ac.id/science/vol27/iss4/4
This Article is brought to you for free and open access by the Universitas Indonesia at UI Scholars Hub. It has been
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Enzymatic Screening and Genotypic Characterization of Thermophilic Bacteria Enzymatic Screening and Genotypic Characterization of Thermophilic Bacteria
from the Hot Springs of Sarawak, Malaysia from the Hot Springs of Sarawak, Malaysia
Authors Authors
Seng Chiew Toh, Samuel Lihan, Sui Sien Leong, Azizul Hakim Lahuri, Wai Cheong Woon, and Wing Woh Ng
This article is available in Makara Journal of Science: https://scholarhub.ui.ac.id/science/vol27/iss4/4
Makara Journal of Science, 27/4 (2023), 279−288
doi: 10.7454/mss.v27i4.1449
279 December 2023 Vol. 27 No. 4
Enzymatic Screening and Genotypic Characterization of Thermophilic
Bacteria from the Hot Springs of Sarawak, Malaysia
Seng Chiew Toh1, Samuel Lihan2, Sui Sien Leong1,3*,
Azizul Hakim Lahuri4, Wai Cheong Woon5, and Wing Woh Ng2
1. Department of Animal Sciences and Fishery, Faculty of Agricultural and Forestry Sciences,
Universiti Putra Malaysia Bintulu Sarawak, Bintulu 97008, Malaysia
2. Institute of Biodiversity and Environmental Conservation, Universiti Malaysia Sarawak,
Kota Samarahan 94300, Malaysia
3. Institute of Ecosystem Science Borneo, Universiti Putra Malaysia Bintulu Sarawak, Bintulu 97008, Malaysia
4. Department of Science and Technology, Faculty of Humanities, Management and Science,
Universiti Putra Malaysia Bintulu Sarawak, Bintulu 97008, Malaysia
5. English Unit (CALC), Universiti Putra Malaysia Bintulu Sarawak, Bintulu 97008, Malaysia
*E-mail: leongsuisien@upm.edu.my
Received October 24, 2022 | Accepted September 16, 2023
Abstract
Owing to their eccentric thermostable ability, thermophiles are among the most utilized extremophiles in various
industries, such as manufacturing, and clinical research. Researchers believe that many unknown thermophiles are yet to
be discovered. This study aimed to genotypically characterize the diversity of thermophiles and screen them for the
potential production of enzymes in the recreational hot springs located at Northwest Coast of Borneo. Water samples
were collected at 45 °C–50 °C from Annah Rais and Panchor hot springs during the sampling period from January 2018
to January 2019. Three samples (water and sediment) were collected twice in a 3-week interval from each pool of the
sampling sites. Each water sample was diluted up to 10−3 and plated on thick nutrient agar at 55 °C for 24 h. Customized
nutrient agar plus Bacto-agar plates were used for the optimum growth analysis of the isolates at 40 °C–90 °C for 24 h.
The thermophiles were isolated, characterized biochemically, and amplified molecularly using DNA fingerprinting and
16S rRNA gene sequencing. Lipase, protease, gelatinase, amylase, catalase, and nitrate reductase enzymatic production
was examined. Twenty-one thermophilic isolates were successfully characterized into seven clusters of Amnoxybacillus
spp. and Geobacillus spp. by studying their phylogenetic dendrograms. Isolates AR10 and AR15 could produce most of
the tested enzymes. All the isolates showed negative results in gelatinase and lipase production. PC14 was the only isolate
that did not produce any of the enzymatic reactions in this experiment. The results showed that most of the thermophiles
isolated from the two Borneo hot springs can synthesize enzymes and have potential to be thermostable. In conclusion,
the search for the thermophilic producers of novel enzymes in Borneo is successful; further research must focus on their
applications.
Keywords: Borneo, enzyme, genotypic, recreational hot springs, thermophiles
Introduction
The unique enzymatic potential of thermophilic and
hyperthermophilic microorganisms was discovered two
decades ago, prompting explorations on their potential
biotechnological, industrial, and clinical applications.
Thermophiles are the extremophiles that optimally grow
in hot environments with temperatures ranging from
45 °C to 122 °C [1]. Thermophiles are grouped in three
major clusters, namely, moderate thermophiles (survives
at temperatures from 55 °C–65 °C), extreme thermophiles
(survives at temperatures from 65 °C–80 °C), and
hyperthermophiles (survives at temperatures higher than
80 °C) [2]. They exhibit normal function and metabolism
in tremendously hot environments. Most thermophiles
are classified as members of the Bacillaceae family,
including Thermobacillus, Coprobacillus, Anoxybacillus,
Sulfobacillus, Halobacillus, Salibacillus, Marinibacillus,
Virgibacillus, Amphibacillus, Alicyclobacillus,
Gracilibacillus, Geobacillus, Jeotgalibacillus,
Breviballicus, Paenibacillus, Aneurinibacillus, and
Ureibacillus, as identified using 16S rRNA gene
sequences [3–5]. Thermophiles are found in habitats such
as hot springs, deep-sea cores, petroleum reservoirs,
deep-sea hydrothermal vents [6], and man-made facilities
such as compost piles, slag heaps, and water heaters [7].
280 Toh, et al.
Makara J. Sci. December 2023 Vol. 27 No. 4
Researchers believe that these thermophiles have
acquired certain thermophilic adaptation abilities, exhibit
high metabolism, and secrete various physically and
chemically stable enzymes [8] with special
biotechnological interest. The thermostability of these
organisms enables them to be utilized as biocatalysts in
most manufacturing industries, such as biomass
deconstruction, fuel-related compound manufacturing,
and leaching or waste management industries.
Thermophilic bacilli are industrially important because
they produce extracellular thermostable enzymes, such as
proteases, amylases, lipases, pullulanase, xylanases, and
others [9]. They can convert surrounding lipids and
carbohydrates to energy and carbon sources. With these
advantages, thermophiles can grow faster than
mesophilic bacteria. Thus, these organisms can speed up
fermentation; reduce microbial contamination risks, high
diffusion rate, or mass turnover; decrease the viscosity of
the liquid medium; and increase the solubility of
polymeric substrates or fats [9]. Thermostable enzymes
are resistant to proteolysis and chemical denaturation.
Although thermophiles have many advantages in
industrial application, they can also be harmful pathogens
isolated from clinical samples and cause serious illness.
A previous study identified two patients infected with
septic meningitis caused by the thermophilic bacteria in
their cerebrospinal fluid [10].
The biodiversity of thermophiles in our tropical habitat
in Borneo has received huge attention from scientists
globally. This study focused on two well-known
recreational hot springs in Borneo, Annah Rais and
Panchor hot springs. Annah Rais hot spring is an ancient
250-year-old hot spring surrounded by mountains and
maintained by the early Bidayuh settlers, along with the
longhouse. It is a natural feature formed by the
geothermal forces heating the underground water to the
surface; the water is drained into Sungai Semadang or
Sungai Sarawak Kiri. It spans 71 km and is about 1 h and
20 min driving distance from the city. Panchor hot
springs is a natural pool surrounded by forests and
situated within them are settled by the Dayak natives. It
spans 50 km and is about a 1h driving journey from the
city. The early native people believed that hot springs are
holy places to seek blessings and cures for various
illnesses. Data profiling on the thermophiles in these hot
springs is still limited and yet to be discovered. This
study aimed to genotypically characterize the diversity of
thermophilic bacteria in the hot springs of the Northwest
Coast of Borneo and screen them for the potential
production of extracellular enzymes.
Materials and Methods
Sampling sites. Water samples were collected from
Annah Rais (latitude 1.1332°N, longitude 110.2676°E)
and Panchor (latitude 1.2540°N, longitude 110.4508°E)
hot springs located in the Northwest Coast of Borneo,
Malaysia from January 2018 to January 2019.
Sample collection and processing. Water samples were
collected from the major heat sources of each hot spring
at different depths as surface water, sediment with 45 cm
depth, sediment with 60 cm depth, and sediment with 120
cm depth. Three samples (water and sediment) were
collected from each pool of the sampling sites and kept
in thermostable flasks for further analysis. Samples were
collected twice in a 3-week interval. The temperature and
pH of different water layers were recorded. Each water
sample was diluted up to 10−3 , plated on thick nutrient
agar (20 g/L) (Merck, Germany) with the pH adjusted
according to the pH level of the hot spring water using 1
M sterile Na-sesquicarbonate solution (Merck,
Germany), and incubated at 55 °C for 24 h. The bacteria
were kept in nutrient broth (OXOID, UK) containing
15% glycerol at −80 °C for further studies.
Biochemical characterization on thermophiles. The
pure culture of each thermophilic bacterial strain was
biochemically identified using Gram staining, sulfur
indole motility (Merck, Germany), phenol red mannitol
salt agar (Merck, Germany), McConkey agar (Merck,
Germany), Simmons citrate agar (Merck, Germany),
triple sugar iron agar (Merck, Germany), and Levine’s
eosin–methylene blue agar (Merck, Germany) tests.
Thermophile DNA extraction and purification. DNA
was extracted from the pure culture of thermophilic
bacterial strains using the cetyl trimethylammonium
bromide (CTAB) DNA extraction method by Sahu et al.
[11] with modification to obtain the maximum yield of
DNA. In brief, 2 mL of the overnight pure culture was
centrifuged at 10,000 rpm for 5 min. The procedure was
repeated twice. The pellet was harvested and resuspended
in 567 µL of Tris ethylenediaminetetraacetic (EDTA)
(Merck, Germany) (TE) buffer, followed by the addition
of 30 µL of 10% sodium dodecyl sulfate (SDS) (Merck,
Germany) and 3 µL of 20 mg·mL−1 proteinase K (Merck,
Germany) to achieve the final concentration of 100
µg·mL−1 proteinase K in 0.5% SDS. The mixture was
then mixed thoroughly and incubated for 1 hour at 37 °C.
After incubation, the mixture was added with 100 µL of
5 M sodium chloride (NaCl) (Merck, Germany) and 80
µL of CTAB/NaCl (Merck, Germany) solution and
incubated at 65 °C for 10 min. Afterward, 780 µL of
chloroform/isoamyl alcohol (Merck, Germany) was
added and centrifuged at 10,000 rpm for 5 min. The
aqueous supernatant was harvested and mixed with an
equal volume of phenol/chloroform/isoamyl (Merck,
Germany) alcohol. The mixture was then mixed well and
centrifuged at 10,000 rpm for 5 min. The supernatant was
harvested again and added with 0.6 mL of isopropanol
(Merck, Germany). DNA was precipitated by spinning
briefly at room temperature. Lastly, the DNA pellet was
washed with 70% ethanol to remove residual CTAB,
Enzymatic Screening and Genotypic Characterization 281
Makara J. Sci. December 2023 Vol. 27 No. 4
centrifuged at 10,000 rpm for 5 min, and resuspended
with 100 µL of TE buffer for further analysis.
DNA fingerprinting. The DNA was amplified by PCR
to determine the strain diversity of the thermophiles using
oligonucleotide probe (GTG)5 (5ʹ-
GTGGYGGYGGYGGYG-3) (Promega, USA) as stated
by Sien et al. [12]. A PCR master mix was prepared with
5 µL of 5X Buffer (Promega, USA), 3 µL of 25 mM
magnesium chloride (MgCl2), 1 µL of 10 mM of
deoxynucleotide triphosphate (dNTP) (Promega, USA),
1 µL of 10 µM (GTG)5 primer (Promega, USA), 9.5 µL
of sterilized distilled water, 5 µL of the isolate’s DNA,
and 0.5 µL of Taq polymerase (Promega, USA).
Amplification was performed using the LabCycler
(SensoQuest GmbH, Germany) with the following PCR
profile: preliminary denaturation at 95 °C for 2 min; 30
cycles of 95 °C for 1 minute, annealing at 50 °C for 1
minute, and an extension at 72 °C for 1 minute; and a
final extension at 72 °C for 5 min. The amplified products
were viewed using 1.2% agarose gel agar in Tris-borate-
EDTA (TBE) (Merck, Germany) buffer at 90 V and 400
mAh for 105 min. The 1 kb DNA ladder (Promega, USA)
was used as the standard DNA size marker for each run.
The DNA fingerprint gel images were then analyzed
using GelJ software Version 2.0. The dendrogram was
constructed to look at the relationship between the
isolates.
16S rRNA sequencing. The identified isolates were
analyzed with 16S rRNA PCR by amplifying their 16S
ribosomal RNA genes [13]. Primers 27F 5ʹ-
AGAGTTTGATCMTGGCTCAG-3ʹ and 519R 5ʹ-
GWATTACCGCGGCKGCTG-3ʹ (Promega, USA) were
used in the PCR amplification. A PCR master mix was
prepared with 10 µL of 5X Buffer (Promega, USA), 6 µL
of 25 mM MgCl2, 3 µL of 10 mM of dNTP (Promega,
USA), 1 µL of each primer (Promega, USA), 8 µL of
sterilized distilled water, 20 µL of the isolate’s DNA, and
1 µL of Taq polymerase (Promega, USA). The
amplification was repeated for 30 cycles with the
following PCR profile: preliminary denaturation step at
95 °C for 10 min, denaturation at 99 °C for 30 seconds,
annealing at 55 °C for 1 min, and extension at 72 °C for
80 seconds, followed by a final extension at 72 °C for 10
min. The samples were cooled at 4 °C. The amplified
products were viewed using 1.0% agarose gel in TBE
buffer at 90 V and 200 mAh for 30 min. The 100 bp DNA
ladder (Promega, USA) was used. The purified PCR
product was sent to Apical Scientific Sdn Bhd (Malaysia)
for further DNA sequencing to identify the closest
identity matches by comparing the product with other
sequences from the data bank in the NCBI website
(http://www.ncbi.nlm.nih.gov) using BLAST.
Optimum temperature determination. The bacterial
isolates were plated on customized nutrient agar plus
Bacto-agar (15 g/L) (Difco Laboratories, USA) plates in
duplicate and incubated at 40 °C, 45 °C, 55 °C, 60 °C,
65 °C, 70 °C, 75 °C, 80 °C, 85 °C, and 90 °C for 24 h.
All colony forming units that grew on the plates were
counted and recorded [14].
Assessment of enzymatic production. Screening for
enzymes such as lipase, protease, gelatinase, amylase,
catalase, and nitrate reductase was performed in the
bacterial cultures grown for 24–48 h at their optimum
temperature. Each enzymatic test was performed in
triplicate. Pseudomonas sp. identified in the laboratory
was used as the control.
Lipolytic enzyme. Lipolytic activity was assessed using
tryptone soy agar (Lab M, United Kingdom)
supplemented with 1% of Tween 20. The presence of a
turbid halo around the inoculum indicated positive
lipolytic activity.
Protease test. Protease activity was assessed using
Mueller–Hinton agar (Himedia, India) supplemented
with 3% of skimmed milk powder. The appearance of a
transparent zone around the inoculum indicated caseinase
activity.
Gelatinase test. Gelatinase activity was assessed by
stabbing the isolate culture onto nutrient agar (Merck,
Germany) supplemented with 3% gelatin. After the
incubation, the medium was cooled at 4 °C for 30 min.
Gelatin liquefaction indicated gelatinase activity.
Amylase test. Amylase activity was assessed using
Difco™ Starch Agar (BD, United States). After staining
with 1% iodine solution (Merck, Germany), the
appearance of a clear halo zone around the colonies
indicated amylase activity.
Catalase test. Catalase test was assessed using hydrogen
peroxide (H2O2) (Merck, Germany). Rapid bubbling
upon dipping the isolates into the H2O2 solution
indicated catalase activity.
Nitrate reductase activity. Nitrate reductase activity
was assessed using Difco™ Nitrate Broth (BD, United
States). Nitrate A reagent containing 0.5 mL of 0.8%
sulfanilic acid in 5N acetic acid and Nitrate B reagent
containing 0.5 mL of 0.6% N, N-dimethyl-alpha-
naphthylamine in 5N acetic acid were added into the
bacteria cultured in nitrate broth. The appearance of a red
color indicated nitrate reductase activity. Meanwhile, a
colorless broth was added with Zn powder to test the
nitrate reductase activity. The presence of a red color
upon the addition of Zn powder indicated the lack of
nitrate reductase activity. If the broth remained colorless
after the addition of Zn powder, then it had nitrate
reductase activity.
282 Toh, et al.
Makara J. Sci. December 2023 Vol. 27 No. 4
Results
Thermophile profiling. The sampling details,
thermophilic bacteria isolates, and their thermal tolerance
properties are stated in Table 1. The water samples
collected from both hot springs had the same moderate
temperature (45 °C–50 °C) but different pH levels. A
total of 50 bacteria was successfully isolated from the hot
springs and grouped into 8% (n = 4) mesophiles (40 °C),
48% (n = 24) thermophiles (55 °C–65 °C), and 44% (n =
22) hyperthermophiles (higher than 80 °C). The optimum
temperature growth of the bacterial isolates was
determined as shown in Figure 1.
Table 1. Thermophilic Bacteria Profiling Details Collected from Annah Rais and Pachor Hot Springs in the Northwest Coast
of Borneo, Malaysia
Sampling
Sites
Code
Sources
Layer of
Water
Temperature
(˚C)
pH
Bacteria
Isolates
Optimum Growth
Temperature
(˚C)
Thermal
Tolerance
Annah
Rais
G
Small pond
Sediment
(45 cm depth)
45
8.63
AR01
AR02
AR03
AR04
60
60
60
90
Thermophile
Thermophile
Thermophile
Hyperthermophiles
J
Small pond
Sediment
(45 cm depth)
45
8.63
AR08
AR09
AR10
AR11
AR18
AR19
60
60
60
90
60
40
Thermophile
Thermophile
Thermophile
Hyperthermophiles
Thermophile
Mesophile
H
Big pond
Surface
Water
45
8.17
AR05
AR06
90
60
Hyperthermophiles
Thermophile
I
Big pond
Sediment
(60 cm depth)
50
8.17
AR07
AR17
90
90
Hyperthermophiles
Hyperthermophiles
K
Big pond
Surface
Water
45
8.17
AR12
AR13
90
90
Hyperthermophiles
Hyperthermophiles
L
Big pond
Sediment
(60 cm depth)
50
8.17
AR14
AR15
AR16
AR20
AR21
60
90
90
90
90
Thermophile
Hyperthermophiles
Hyperthermophiles
Hyperthermophiles
Hyperthermophiles
Panchor
A
Pond
Surface
Water
45
6.62
PC01
PC02
PC03
PC15
90
90
90
60
Hyperthermophiles
Hyperthermophiles
Hyperthermophiles
Thermophile
B
Pond
Sediment
(45 cm depth)
47
6.62
PC04
PC05
PC06
PC07
PC08
PC16
PC29
60
40
40
40
90
90
90
Thermophile
Mesophile
Mesophile
Mesophile
Hyperthermophiles
Hyperthermophiles
Hyperthermophiles
C
Pond
Sediment
(120 cm depth)
50
6.62
PC09
PC17
PC18
90
60
60
Hyperthermophiles
Thermophile
Thermophile
D
Pond
Surface
Water
45
6.62
PC10
PC19
PC20
PC21
PC22
60
60
60
60
60
Thermophile
Thermophile
Thermophile
Thermophile
Thermophile
Enzymatic Screening and Genotypic Characterization 283
Makara J. Sci. December 2023 Vol. 27 No. 4
Table 1. Continue
Figure 1. Optimum Temperature Growth of the Thermophiles Isolated from Annah Rais and Panchor Hot Springs in the
Northwest Coast of Borneo, Malaysia
Phylogenetic analysis and thermophile identification.
The gel electrophoresis image of the (GTG)5
fingerprinting analysis and the constructed phylogeny
tree are demonstrated in Figures 2 and 3. All the isolates
were clustered into seven groups. Based on the analysis
of colony morphology and phylogenetic tree, one isolate
from each group (AR05, AR15, PC06, AR16, AR10,
PC01, and PC14) was chosen for further characterization
by amplifying its 16S rRNA gene sequence. The
amplified DNA product was detected at 600 bp.
Sequence homology was observed between the partial
sequences of A. rupiensis strains 1–35 and isolate PC14
and between G. stearothermophilus strain WSUCF-035C
and isolate AR10 in the NCBI Genbank database.
BLAST sequencing result revealed that most of the
isolated thermophiles fall in two main genera (Table 2).
Assessment of biochemical and enzymatic
production. The seven isolates were biochemically
characterized and tested for the production of several
enzymes (Table 3). All the bacteria were Gram-positive
and had rod shapes. All the seven isolates tested negative
on all the selective and differential agars and were unable
to produce gelatinase and lipase enzyme. PC14 was the
only isolate that did not produce any enzymatic reaction
and showed negative results on all the tested enzyme
activities. AR10 and AR15 showed positive results in
most of the tested enzyme activities except gelatinase and
lipase activity. The isolates from the Borneo hot springs
were able to synthesize most of the enzymes with high
thermostability.
Sampling
Sites
Code
Sources
Layer of
Water
Temperature
(˚C)
pH
Bacteria
Isolates
Optimum Growth
Temperature
(˚C)
Thermal
Tolerance
E
Pond
Sediment
(45 cm depth)
47
6.62
PC11
PC12
PC13
PC23
PC24
60
60
60
90
60
Thermophile
Thermophile
Thermophile
Hyperthermophiles
Thermophile
F
Pond
Sediment
(120 cm depth)
50
6.62
PC14
PC25
PC26
PC27
PC28
90
90
60
60
90
Hyperthermophiles
Hyperthermophiles
Thermophile
Thermophile
Hyperthermophiles
284 Toh, et al.
Makara J. Sci. December 2023 Vol. 27 No. 4
Figure 2. Gel Image Showing the (GTG)5 Fingerprinting DNA Amplification Products of Eleven Isolates. The PCR Products
were Loaded Onto 1.2 % (w/v) Agarose Gel and Run at 90 V for 105 min. M 1kb DNA Ladder. 1–12 Selected
Thermophiles Isolates, Namely, AR02, AR03, AR05, AR07, AR08, AR09, AR10, AR11, AR12, AR13, and AR16. NC
Negative Control
Figure 3. Phylogenetic Tree Constructed using GelJ Software Version 2.0 Showing the Relationships of the Thermophiles
Isolated from the Hot Springs of the Northwest Coast of Borneo. Comments Showed All Thermophile Isolates
Table 2. Isolated Thermophiles Isolated from the Hot Springs of the Northwest Coast of Borneo
Isolates
Identification
Gene bank access
number
Closest related species
Similarity
%
PC01
Anoxybacillus rupiensis
MF037808
A. rupiensis strain T7
100
PC06
Anoxybacillus flavithermus
MH005096
A. flavithermus strain SS6_1017_022
100
PC14
Anoxybacillus rupiensis
KF583689
A. rupiensis strain 1-35
100
AR05
Anoxybacillus gonensis
MH005096
A. gonensis strain Lhs-8
100
AR10
Geobacillus stearothermophilus
MF965163
G. stearothermophilus strain WSUCF-035C
100
AR15
Geobacillus stearothermophilus
KP338613
G. stearothermophilus strain IR4
100
AR16
Geobacillus stearothermophilus
CP016552
G. stearothermophilus strain DSM 458
100
Enzymatic Screening and Genotypic Characterization 285
Makara J. Sci. December 2023 Vol. 27 No. 4
Table 3. Biochemical Characteristics of the Isolates Selected Due to Their Potential as Enzyme Producers
Isolates
Biochemical
Enzymatic
Gram staining
Morphology
TSI
Citrate
SIM
EMBA
MacConkey
MSA
Lipase
Protease
Gelatinase
Amylase
Catalase
Nitrate reductase
PC01
+
Rod
−
−
−
−
−
−
−
−
−
+
+
−
PC06
+
Rod
−
−
−
−
−
−
−
−
−
−
+
+
PC14
+
Rod
−
−
−
−
−
−
−
−
−
−
−
−
AR05
+
Rod
−
−
−
−
−
−
−
−
−
−
−
+
AR10
+
Rod
−
−
−
−
−
−
−
+
−
+
+
+
AR15
+
Rod
−
−
−
−
−
−
−
+
−
+
+
+
AR16
+
Rod
−
−
−
−
−
−
−
−
−
−
−
+
Con-
trol
+
+
+
−
−
+
Note: − absent; + present; Control: Pseudomonas sp.
Discussion
The profile and diversity of thermophilic bacterial
communities in the two recreational hot springs
distributed in the Northwest Coast of Borneo were
investigated (Table 1). Our study categorized the bacteria
into 8% mesophiles, 48% thermophiles, and 44%
hyperthermophiles. Temperature is an important growth
parameter for bacterial diversity [15], and these two
factors exhibit a positive linear correlation [16]. An
increase in temperature, especially in harsh environments
such as hot springs, remarkably decreased bacterial
diversity and species richness, allowing only dominant
extremophiles to grow. Fingerprinting analysis clustered
the thermophiles into seven bacterial groups based on
their genetic closeness. Based on the colony morphology
and phylogenetic tree analysis, one isolate from each
phylogenetic group was identified using 16S rRNA gene
sequence. The thermophiles isolated from the hot springs
in Borneo were unique. They could grow in the water and
sediment of the hot springs at moderate temperature (45
°C–50 °C). However, as the optimum growth
temperature for each isolate was reported high, they
possess a substantially potential for thermostable enzyme
production. They can be used in industrial processes,
such as the degradation of environmental pollutants.
BLAST sequencing result revealed that most of the
isolated thermophiles fall in two main genera, i.e.,
Anoxybacillus spp. and Geobacillus spp. (Table 2).
Anoxybacillus spp. and Geobacillus spp. are thermophiles
that can survive and abundantly multiply in these high-
temperature hot springs. In 2000, Anoxybacillus spp. was
first isolated from manure samples and categorized as
anaerobic, alkaliphilic, fermentative, moderately
thermophilic, endospore-forming rod-shaped bacteria
[17]. Our findings showed that Anoxybacillus spp. are
found in the alkaline and acidic hot springs of Borneo and
concurred with the study of Goh et al. [18], who
explained that Anoxybacillus spp. are well adapted to the
environmental pH. Isolates PC01 and AR05, both
detected as Anoxybacillus spp., can grow in temperatures
up to 90 °C, although most studies [8, 17, 18] have
described their optimum growth to be 65 °C. Geobacillus
spp. are endospore-forming obligate rod-shaped
thermophiles that can grow in temperatures up to 80 °C
[19]. Both thermophiles are widely isolated found the hot
springs. We detected Anoxybacillus flavithermus, which
was also successfully isolated by Caspers et al. [20] from
the dairy-processing plants in New Zealand and the
Netherlands, and Anoxybacillus rupiensis, which was
first discovered by Derekova et al. [21] in 2007 as a novel
thermophilic bacterium isolated from Rupi Basin,
Bulgaria. Similar results were obtained by Chai et al.
[22], who successfully isolated Anoxybacillus spp. from
Klah River and Dusun hot springs in Malaysia. Most of
the isolated thermophiles have vast potential for
industrial and agricultural functions. Isolate AR05,
identified as Anoxybacillus gonensis, can secrete amylase
enzyme for poly-3-hydroxybutyrate degradation [23].
Beris et al. [24] believed that the G2ALT gene in
Anoxybacillus spp. in PP-loop ATPase superfamily may
be responsible for aluminum tolerance. Kambourova et
al. [25] stated that A. flavithermus is widely used in
agriculture to produce a thermostable multienzyme
xylanase complex in the hemicellulosic fraction of
terrestrial plants to improve the yield of sugar production
from oat-spelt xylan. Neumann et al. [26] stated that
286 Toh, et al.
Makara J. Sci. December 2023 Vol. 27 No. 4
Geobacillus stearothermophilus, a hyperthermophile, is
often applied in sterilization because it can survive after
the process in autoclaves. Similar results were obtained
by Sharma et al. [27], who found 13 Geobacillus spp.
isolated from the Soldhar hot spring site of Garhwal
Himalaya in India. Geobacillus spp. has a broad spectrum
of carbohydrate-utilization ability, which is yet to be
discovered because this bacterium is still new to
scientific research.
Among the thermophiles, the genera Geobacillus and
Amoxybacillus have received considerable attention due
to their thermostable adaptation and potential applications
in producing industrially important enzymes. Hot springs
are considered harsh environments with low nutritional
status and sometimes high mineral content. Only the
toughest extremophiles can endure these conditions for
growth. The natural selection of most extremophiles
varies with different ecological habitats. Thus, the
thermophile population in most hot springs is dominated
mainly by bacilli, including Geobacillus spp. and
Amoxybacillus spp. On the basis of the results, all seven
isolates were unable to produce gelatinase and lipase
enzymes. Thus, most of the bacilli were unable to use
gelatin and oil as their food sources. However, most of
the reported thermophiles are lipase and gelatinase
producers [28]. Pimpliskar et al. [29] detected gelatinase
enzyme in the thermophiles isolated from the hot water
in Vajreshwari, India. Most of the isolates showed
positive nitrate reductase activity except PC01 and PC14,
which are Anoxybacillus rupiensis. The nitrate reductase
enzyme is responsible for nitrogen assimilation in most
plants because it provides an enzymatic source of nitric
oxide. AR10 and AR15 were identified as G.
stearothermophilus, which produces most of the tested
enzymes including amylase, catalase, protease, and
nitrate reductase. However, AR16 was also identified as
G. stearothermophilus but did not show as much
enzymatic activity as AR10 and AR15 possibly due to the
different adaptations of different bacterial strains. In
addition, G. stearothermophilus can produce thermostable
and thermoactive α-amylase [30], oxidase, catalase [31],
and highly thermostable protease [32].
Conclusions
Our study reported 4 mesophiles, 22 thermophiles, and
24 hyperthermophiles with seven clusters of
Amnoxybacillus spp. and Geobacillus spp. isolates from
the two hot springs. Isolates AR10 and AR15 could
produce most of the tested enzymes except gelatinase and
lipase. We believe that other potential thermophiles are
yet to be identified. Thus, further studies are crucial to
discover the full potential of the thermophiles in the
industrial and commercial sectors. A. flavithermus
isolated in this study can enhance the sugar production
yield by facilitating the enzymatic hydrolysis of xylan.
Meanwhile, A. salavatliensis can produce alpha-
glucosidase, the catalyst used in starch hydrolysis that
may aid in the digestion of dietary carbohydrates in the
food industry.
Acknowledgement
This research study was supported by the grant awarded
by Putra Grant (9692700). We are grateful to the
supporting laboratory staffs of virology laboratory and
microbiology laboratory, Universiti Malaysia Sarawak
where the laboratory analysis was conducted.
Conflict of Interest Disclosure
None declared.
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